Agents that activate muscarinic acetylcholine receptors (mAChRs) have exciting potential as novel treatments for positive symptoms, negative symptoms, and cognitive disturbances in patients suffering from schizophrenia. Previous mAChR agonists failed in clinical development due to a lack of selectivity for individual mAChR subtypes and adverse effects associated with activation of peripheral mAChRs. We have now discovered and characterized highly selective positive allosteric modulators (PAMs) for individual mAChR subtypes and found that selective PAMs for the M4 subtype have antipsychotic-like effects in animal models and that M1-selective PAMs have robust cognition-enhancing effects and may have actions that predict reduction in negative symptoms. Interestingly, M4 PAMs have effects in animal models that suggest that these agents act by reducing signaling by dopamine (DA), a neuromodulator that is known to be important for the positive symptoms associated with schizophrenia. We found that M4 PAMs reduce evoked DA release in the midbrain, suggesting that these agents act in part by inhibiting release of DA from presynaptic DA terminals. However, M4 is not present in midbrain DA nerve terminals, suggesting that M4 PAMs could not act directly in presynaptic terminals to inhibit DA release. In addition to modulating DA release, we also present data suggesting that M4 PAMs can directly inhibit responses to administration of selective agonists of the D1 DA receptor D1R. Since D1R agonists bypass endogenous DA, this suggests that M4 PAMs must also act downstream of inhibition of DA release. Interestingly, the large majority of M4 in the striatum and nucleus accumbens (NAc) is expressed in striatal projection neurons (SPNs), which are the primary projection neurons of the striatum and NAc and the primary target for DA nerve terminals in these regions. More specifically, M4 is selectively expressed in a subpopulation of SPNs that also express the D1R (D1-SPNs). Thus, we will perform a series of studies to evaluate effects of M4 PAMs on D1-SPNs that would reduce DA signaling. First we will test the hypothesis that activation of M4 in D1-SPNs induces release of a local messenger that exits the cell and activates CB2 cannabinoid receptors on neighboring DA terminals to inhibit DA release. Second, we will test the hypothesis that M4 activation can also directly oppose postsynaptic effects of DA in SPNs by activation of G?i/o G-proteins which inhibit D1R-mediated activation of a unique G protein termed G?olf and subsequent activation of adenylyl cyclase and downstream signaling pathways. In contrast to M4, extensive studies suggest that M1 may act in the prefrontal cortex to reduce cognitive disturbances and negative symptoms associated with schizophrenia. Thus, we will also test the hypothesis that selective M1 PAMs act in the prefrontal cortex to reverse deficits in synaptic plasticity between the hippocampus and prefrontal cortex and reverse specific cognitive deficits and negative symptoms that are observed in rodent models of schizophrenia.